Pathogenic infections, in particular fungal infections, of animals (including humans and agricultural and domestic animals), and plants, (in particular, agriculturally-significant plants) produce significant losses in productive capacity worldwide.
True fungal pathogens may be classified into at least three major classes, the Phycomycetes, the Ascomycetes, and the Basidiomycetes.
With particular regard to fungal pathogens which infect humans and other mammals, over 40 species are known which attack epithelial tissues (eg. hair, skin, and nails), causing diseases and discomforts of an annoying nature, such as, for example, tinea pedis (athlete's foot), tinea cruris, tinea corporis (ringworm) due to infection by the dermatophytes Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum, and Microsporum canis; candidiasis due to Candida albicans, including cutaneous candidiasis (thrush), onychia, paronychia, external genital candidiasis, candidal balanitis; pityriasis versicolor due to Pityrosporum orbiculare (Malassezia furfur); and jockey-strap itch. Additionally, certain species of fungi infect sub-cutaneous tissues, including those of major internal organs, to produce serious systemic diseases, including blastomycosis, coccidiomycosis, histoplasmosis, and sporotrichosis. These more serious fungal infections are generally soil-borne, easily aerosolized, spread by air currents, and, as a consequence, contracted by inhalation. The fungal pathogens are mostly dimorphic, occurring as a mycelium in the non-pathogenic state, which bears infectious spores.
Many more fungal pathogens produce a variety of diseases in plants, wherein different species of fungi tend invade particular species, and particular tissues, of plants. Crop damage from fungal infection accounts for many millions of dollars in lost profits annually. The Basidiomycetes are of particular importance in agriculture, and include the rust fungi, such as, for example, Puccinia spp., Cronartium ribicola, and Gymnosporangium juniperi-virginianae; the smut fungi, such as, for example, Ustilago spp., which infect corn and oats, amongst others, causing up to 30% losses annually. Additionally, take-all disease, which is caused by infection of wheat plants by the fungal pathogen Gaeumannomyces graminis var tritici (or commonly known as the “take-all” fungus) is the most significant root disease of wheat around the world and currently leads to 10% loss of the annual wheat crop in Australia (Murray and Brown, 1987). Other important fungal diseases in plants include crown wart of alfalfa disease, which is caused by Physoderma alfalfae; bitter rot of apple disease, caused by Glomerella cingulata; apple rust, caused by Gymnosporangium juniperi-virginianae; apple scab, caused by Venturia inaequalis; banana wilt, caused by Fusarium oxysporum f. cubense; loose smut of barley, caused by Ustilago nuda Rostr.; early and late blight in celery, caused by Septoria apiicola; Fusarium yellow in celery, caused by Fusarium oxysporum f. apii Snyder Hansen; ergot in grain crops, and grasses, caused by Claviceps purpurea; stem rusts, caused by Puccinia spp., in particular P. graminis; late blight in potato, caused by Phytopthera infestans; and citrus root rot diseases caused by Armillaria mellae. However, this list is not exhaustive.
Methods for the prophylactic and/or therapeutic treatment of fungal and bacterial infections in animals and plants generally involve the application of anti-fungal and anti-bacterial chemicals; the use of biocontrol agents; and, more recently, and particularly in the case of plants, the genetic engineering of crops to express disease tolerance or disease-resistance genes therein.
Funcidal and Fungistatic Chemical Compounds:
Anti-fungal chemicals are varied in composition and designed to either eradicate the fungal pathogen, such as, for example, by acting against the fungal spores, or alternatively, to prevent the germination of fungal spores once they have infected their host. However, most chemicals do not fall exclusively into a single category. For example, elemental sulfur has been used to protect apple crops against apple scab, however the same chemical is eradicative when used against a rust fungus.
A wide variety of pyridine compounds and derivatives thereof having varying, and often multiple, action, are used in agrochemicals and pharmaceuticals against fungal pathogens, such as, for example, 3-(2-methylpiperidino) propyl-3,4-dichlorobenzoate; cephalosporin C; cephapirin sodium; pyrithione zinc (i.e. 2-mercaptopyridine N-oxide); and 2-sulfanylamidopyridine.
Many of the compounds used commonly to treat fungal infections in humans interfere with fungal sterol biosynthesis. For example, the imidazoles (including bifonazole [i.e. 1-(α-biphenyl-4-ylbenzyl)-imidazole], clotrimazole, econazole nitrate, and miconazole nitrate [i.e. 1-[2,4-dichloro-β-(2,4-dichlorobenzyloxy)phenethyl]imidazole nitrate]), which have broad spectrum antifungal activity against dermatophytes and yeasts alter fungal cell membranes by interfering with ergosterol production. The allylamines, such as, for example, terbafine hydrochloride (C21H25 N. HCl), are also of use in the treatment of infections by dermatophytes, also interfere with ergosterol production. Terbafine also results in the accumulation of squalene in the fungus, resulting in fungal cell death. Amorolfine hydrochloride (cis-4-[(RS)-3-[4-(1,1-dimethylpropyl)phenyl]-2-methyl propyl]-2,6-dimethyl morpholine hydrochloride) is in a relatively new class of compounds active against a wide range of yeasts, dermatophytes, moulds, and dimorphic fungi. The fungicidal/fungistatic effect of amorolfine hydrochloride is based upon modification to the fungal cell membranewhich is produced by reducing the ergosterol content and increasing the levels of sterically-nonplanar sterols in the fungal cell membrane.
Agricultural fungicides and their modes of application are reviewed in detail by Gennaro et al. (1990), and Kirk-Othmer (1980). These compounds are generally of the class of polysulfides; heavy-metal fungicides; and the organic fungicides, such as, for example, the quinones (in particular, chlroanil and dichlone), organic sulfur-containing compounds (in particular, the dithiocarbamates), imidazolines and guanines (in particular heptadecyl-2-imidozolinium acetate; and dodecylguanidium acetate), trichloromethylthiocarboximides [in particular, N-trichloromethylthio)4-cyclohexene-1,2-dicarboximide (captan); and N-(trichloromethylthio) phthalimide (folpet)], the chlorinated and/or nitrated benzene derivatives [in particular, 2,3,5,6-tetrachloronitrobenzene; pentachloronitrobenzene (PCNB); 1,3,5-trichloro-2,4,6-trinitrobenzene; 1,2,4-trichloro-3,5-dinitrobenzene; hexachlorobenzene; 2,6-dichloro-4-nitroaniline (dichloran); 1,4-dichloro-2,5-dimethoxybenzene; and tetrachloroisophthalonitrile (chlorothalonil)]. Various other compounds having systemic anti-fungal activity in agricultural applications (i.e. systemic fungicides) include the oxathiins (in particular, carboxin); benzimidazoles [in particular, methyl-2-benzimidazolylcarbamate (MBC)]; pyrimidines [in particular, 5-butyl-2-dimethylamino-6-methyl-4(1H)-pyrimidinone (dimethirimol); the 2-ethylamino analogue, ethirimol; and α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol (triarimol)]. The antibiotics, including cycloheximide, bastocidin S, kasugamycin, and the polyoxins, are also used extensively.
In most cases the mode of action of known agricultural anti-fungal compounds remains elusive, and, as with antifungal compounds that are effective against fungal pathogens of humans, many compounds alter fungal cell membrane metabolism. For example, triarimol and related compounds inhibit steps in sterol biosynthesis. However, carboxin is known to block mitochondrial respiration by inhibiting succinate dehydrogenase, whilst the benzimidazoles disrupt cell structures, such as those required for mitosis.
Anti-fungal Biocontrol Agents:
Biological control protection refers to the introduction of living organisms, such as, for example, bacteria, fungi, and insects, to control plant pathogens. Three properties make biocontrol agents a desirable option in plant protection against fungal pathogens. First, biocontrol agents are generally natural products and, as such, are less likely to have a detrimental effect on the environment than synthetic chemicals. Secondly, biocontrol agents are relatively inexpensive compared to synthetic chemicals. Third, biocontrol agents represent an inexhaustible source of protectant, because microorganisms can be maintained in culture, and expanded rapidly and inexpensively.
Biocontrol agents either act by occupying the site of infection of a pathogen, and competing with the pathogen for nutrients derived from the plant, or alternatively, the biocontrol agent produces fungicidal and/or fungistatic compounds that specifically attack the fungal pathogen. Significant factors important in biological control (such as colonization, antibiosis, siderophore production etc.) have been identified, however the influence of these factors varies with environmental conditions so it is difficult to assign a universal mechanism of action to biocontrol agents (Weller, 1988).
The universal feature of all biocontrol agents is their capacity to rapidly multiply in the cells and tissues that are infected by the pathogenic agent and to form an association therewith, a process which has been termed “colonization” (Suslow, 1982). In fact, Weller (1988) identified three factors as being responsible for the suppressive nature of various bacteria against fungi: colonization of the root; the production of fungicides and/or fungistatic compounds, including antibiotics, HCN (Fravel, 1988), or hydrogen peroxide (Wu et al, 1995; U.S. Pat. No. 5,516,671), amongst others; and the production of fluorescent siderophores, or high-affinity iron-transport agents. In this regard, the different colonization host range shown by biocontrol bacteria (Weller, 1988) suggests there is some host specificity in colonization, and that a number of factors are involved. As will be known to those skilled in the art, there is also host-specificity in the action of fungicides, and this might explain why some strains are ineffective against certain pathogens (Schroth and Hancock, 1981). Additionally, not all biocontrol agents act by producing fungicides or fungistatic compounds (Kraus and Loper, 1992).
There are a large number of systems in which biocontrol is effective (Weller, 1988). In general, biocontrol agents have been shown to have a beneficial effect to the plant under controlled conditions in the glasshouse and in a large number of cases in the field (Baker and Cook, 1974). There are currently numerous biocontrol agents used by farmers for disease control (Schroth and Hancock, 1981), showing that it is effective and viable as a method to control plant diseases in the field.
For example, Sclerotinia rot, caused by the fungi Sclerotinia sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum, is one of the most destructive diseases of plants, affecting over 380 ornamentals (e.g. aster, begonia, calendula, chrysanthemum, fuchsia, gerbera, lupin, pelargonium, and petunia), field crops (e.g. alfalfa, canola, dry bean, hemp, lentil, oilseed rape, peanut, potato, red clover, safflower, soybean, sunflower, sweetclover, and tobacco), vegetables and fruits (e.g. artichoke, asparagus, avocado, bean, broccoli, cabbage, carrot, celery, chickpea, chicory, cucumber, eggplant, endive, fennel, kiwi fruit, leek, lettuce, parsley, pea, pepper (chilli, red or sweet), snap bean, tomato, watermelon, garlic and onion) and herbs (e.g. coriander, chives, dill, fennel, and wintercress). A biological plant-protection agent, containing as an active ingredient contains viable spores of the soil fungus Coniothyrium minitans, has been developed recently which has specific antagonistic action against the survival structures (sclerotia) of these fungal pathogenic agents. Once applied and incorporated into the soil, C. minitans germinates and attacks the sclerotia (resting survival structures) of the pathogens within the soil, thereby reducing recurrences of the disease in the soil.
In the case of take-all disease in wheat, Pseudomonas sp., have been identified which produce a low molecular weight siderophore, sometimes fluorescent, capable of complexing and actively-transporting iron inside the cell, to produce an iron deficiency in the soil (Buyer and Leong, 1986; Leong, 1986), thereby effectively starving the fungal pathogen of soil-derived iron necessary for germination and growth. However, siderophore production is now thought only to be important in the biocontrol of take-all disease in alkaline soils which have low iron concentration, wherein iron may be a limiting nutrient for the fungus (Kloepper et al., 1980). Moreover, Fravel, (1988); Hamdan et al. (1991); and Thomashow et al., (1990), have suggested that the dominant important factor in disease suppression is the production of fungicides and/or fungistatic compounds by Pseudomonas Sp., in particular phenazine-1-carboxylic acid (Thomashow et al., 1993); and 2,4-diacetylphloroglucinol, a normal intermediary in a pathway in bacteria which inhibits a range of fungal pathogens, and is found in a wide range of Pseudomonads (Keel et al., 1992; 1996). Phenazine has been extensively characterised in take-all biological control protection, however the effectiveness of 2,4-diacetylphloroglucinol against take-all has only been partially characterised (Raaijmakers and Weller, 1998). Pseudomonas fluorescens strain CHA0 has been also extensively studied and shown to produce an effective amount of 2,4-diacetylphloroglucinol for the suppression of black rot disease of tobacco and take-all disease of wheat (Laville et al., 1992).
Additionally, the isolated Pseudomonas strain AN5, has been shown to have a wide host range in so far as it is able to colonize the roots of a number of plant species, and is an effective biocontrol agent against take-all disease in agar plate assays, pot experiments, and in field trials (Nayudu et al. 1994b). Those authors concurred with Thomashow et al., (1993) in concluding that the dominant effects of this bacterium appeared to reside in the production of fungicides and/or fungistatic compounds, rather than in the production of siderophores.
Although other biocontrol agents have been tested for their ability to control take-all disease, such as, for example, non-pathogenic strains of G. graminis var. graminis (Wong et al., 1996), they are not a feasible method for large scale control of take-all as there is no current technology available to grow such fungi on a large scale. The cost of producing such a fungal agent and to apply same in the field is prohibitively high compared to bacterial biocontrol agents.
Genetically-manipulated Plants Expressing Disease Resistance or Tolerance:
In certain instances of disease in plants, genes which encode particular enzymes that are involved in the production of anti-fungal (i.e. fungicidal and/or fungistatic) compounds have been identified and expressed in plants to introduce tolerance or resistance thereto. In such cases, there is a requirement for the plant to be capable of expressing the introduced gene(s) and to produce the anti-fungal product from either endogenous plant metabolites, or alternatively or in addition, from exogenous substrates. As will be known to those skilled in the art, once synthesized, the anti-fungal compound must be capable of diffusion to an appropriate site of action, or actively transported, to exert its action against the invading pathogen.
One example of such protection is the expression of a gene encoding the Aspergillus niger or Talaromyces flavus glucose oxidase enzymes (EC 1.1.3.4) to control Phytopthera infestans, or Veticillium dahliae (Murray et al., 1997; Stosz et al., 1996; Wu et al., 1995; U.S. Pat. No. 5,516,671) in plants. The efficacy of this approach was based upon the involvement of hydrogen peroxide in plant defense responses in incompatible plant-pathogen interactions, wherein it may activate the production of phytoalexins and other cellular protectants, such as, for example, salicylic acid and glutathione-5-transferases; induces cross-linking of hydroxyproline-rich glycoproteins (HPGP); and is involved in triggering hypersensitive cell death in response to pathogen invasion. Additionally, hydrogen peroxide is a product of the enzymic action of glucose oxidase, which catalyzes the oxidation of glucose to δ-gluconolactone and hydrogen peroxide, and, as a consequence, the expression of glucose oxidase in plants was considered a suitable means for producing hydrogen peroxide as an anti-fungal agent. The attractiveness of this approach arose, in part, from the knowledge that the glucose oxidase gene of Penecillium dangearii, is involved in the effective biocontrol of V. dahliae by this organism (Kim et al., 1988, 1990; U.S. Pat. No. 5,516,671).
Plants expressing a range of leucine-rich repeat proteins that comprise nucleotide binding sites have also been described as providing improved protection against various diseases in plants, including rust fungi (see, for example, International Patent Application No. PCT/AU95/00240).